Mechanistic Elucidation Guided by Covalent Inhibitors for the Development of Anti-diabetic PPARγ Ligands

Abstract

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-regulated transcription factor that plays crucial roles in adipogenesis, lipid metabolism, and glucose homeostasis. There are several PPARγ ligands having anti-diabetic activity and they commonly inhibit phosphorylation of PPARγ at Serine 273 (Ser273). Recently reported PPARγ ligand SR1664, which selectively block the phosphorylation with no classical agonism, has potent anti-diabetic activity, indicating that the inhibition of Ser273 phosphorylation is sufficient to provoke anti-diabetic effects. In this study, we revealed the x-ray structure of PPARγ co-crystallized with SR1664 bound to the alternate binding site of PPARγ, and we confirmed that this binding directly blocks phosphorylation of Ser273. Furthermore, by using synthesized covalent inhibitors as chemical tools, we demonstrated that the inhibition of phosphorylation is attributed to the occupation of a specific site which is a hydrophobic region between Helix 3 (H3) and β3–β4 at the binding pocket of PPARγ. In high-fat diet-induced obese mice, we confirmed anti-diabetic activity of a covalent inhibitor which is rationally designed to bind at the specific site in PPARγ to block phosphorylation of Ser273. Lastly, the target selectivity of the covalent inhibitor was demonstrated by fluorescence-based visualization of target proteins complexed with its covalent probe containing a bioorthogonal functional group.